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Kinetic Analysis of Truncation and Elongation Mutants of the CapG Severing Mutant

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PAGE 1

KINETIC ANALYSIS OF TRUNCATI ON AND ELONGATION MUTANTS OF THE CapG SEVERING MUTANT By ANDREA ROEBUCK A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Andrea Roebuck

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To my mother, whose presence here today exemplifies perseverance.

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iv ACKNOWLEDGMENTS I would first like to extend my thanks to my mentor (whose patience is great) and to my supervisory committee for their thought ful input. Members of my lab should be commended for their support. I thank my fa mily for their encouragement and love.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii LIST OF ABBREVIATIONS............................................................................................ix ABSTRACT....................................................................................................................... ..x CHAPTER 1 INTRODUCTION........................................................................................................1 Actin.......................................................................................................................... ...1 Actin-Regulatory Proteins............................................................................................2 Origins of the Name CapG...........................................................................................3 Characterizing CapG....................................................................................................3 Evolution/Sequence Comparison..................................................................................4 CapG Sequence, Structure and Function......................................................................4 Regulation of CapG......................................................................................................5 Cellular Distribution.....................................................................................................6 Membrane Ruffling......................................................................................................6 Mutations to CapG........................................................................................................6 Severing Mutant Mutations..........................................................................................7 2 MATERIALS AND METHODS.................................................................................9 Site-Directed Mutagenesis............................................................................................9 Protein Expression and Purification.............................................................................9 Actin Purification........................................................................................................10 Kinetic Assays............................................................................................................11 Subcritical Actin Monomer Fluorescence Assay................................................11 Monomer Sequestration Assay............................................................................12 Capping Assay.....................................................................................................12 Severing Assay....................................................................................................12

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vi 3 RESULTS...................................................................................................................14 Purification of CapG Mutants.....................................................................................14 Structure-Function Analysis.......................................................................................14 Effects of Actin Monomer Binding by CapG Mutants.......................................14 Capping................................................................................................................16 Severing...............................................................................................................17 4 DISCUSSION.............................................................................................................21 Role of the S1-S2 Linker Length on Function............................................................21 Structural Determinants of Capping and Severing.....................................................21 Comparison between CapG Severi ng Mutant and New Mutants...............................23 5 CONCLUSION...........................................................................................................28 LIST OF REFERENCES...................................................................................................29 BIOGRAPHICAL SKETCH.............................................................................................32

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vii LIST OF TABLES Table page 2-1. Primer design for Ca pG severing mutants.................................................................13 4-1. Functional activities of CapG and its mutants............................................................24

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viii LIST OF FIGURES Figure page 1-1 Sequence analysis of CapG, gels olin, villin, fragmin, and severin............................8 3-1 Protein gels of the CapG severi ng mutant and its triplicate mutants.......................17 3-2 Monomer Binding of CapG Severing Mutant and its triplicate mutants.................18 3-3 Capping activities of CapG Seve ring Mutant triplicate mutants..............................19 3-4 CapG Severing Mutant triplicate mutants lack severing activity.............................20 4-1 Structure of the CapG severing mutant....................................................................25 4-2 Possible model of filament capping by gelsolin.......................................................26 4-3 Sequence of events during severing of actin by fully activated gelsolin.................27

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ix LIST OF ABBREVIATIONS Angstrom ATP Adenosine 5’-triphosphate C Degree Celsius cDNA Complementary deoxyribonucleic acid Cys Cysteine DNA deoxyribonucleic acid DEAE diethylaminoethyl DTT dithiothreitol EDTA ethylenediaminetetraacetic acid EGTA [ethylenebis(oxyeth ylenenitrilo)] tetraacetic acid g gravity h hour IPTG isopropyl-1-thio-D-galactoside kb kilobase kDa kilodalton em wavelength of emission ex wavelength of excitation L liter LB Luria-Bertani broth micro m meter min minute M molar n nano PCR polymerase chain reaction pH hydrogen ion concentration pyrene N -(1-pyrenyl) iodoacetamide SDS sodium dodecyl sulfate Tris tris(hydroxymethyl)aminomethane

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x Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science KINETIC ANALYSIS OF TR UNCATION AND ELONGATION MUTANTS OF THE CapG SEVERING MUTANT By Andrea Roebuck May 2006 Chair: Frederick Southwick Major Department: Medical Sciences —Molecular Genetics and Microbiology Numerous proteins help re gulate the actin cytoskelet on network, one of several systems that necessitate cellular movement. One such regulator is CapG, whose main role is to cap the barbed ends of actin f ilaments, thereby preventing actin monomers from additional polymerization at the barbed end. Other family members in the gelsolin superfamily (to which CapG belongs) possess the ability to sever actin filaments. CapG was mutated in two separate locations ba sed on family-member sequence homology, to create a mutant that could both sever and cap filamentous actin (the CapG severing mutant). In this work, site-directed mutage nesis was employed to change the critical domain I-II linker region of the CapG se vering mutant by three amino acids. One insertion and two deletion mutants were created. The three mutants (CapG severing mutant +AAA, –KHV, and –GGV) were then subject to three types of kinetic assessme nts: monomer binding, capping, and severing of pyrene-labeled actin. The monomer binding curve of CapG severing mutant +AAA was

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xi most similar to its parent CapG severing mutant. However its affinity for actin monomers and for mutants –KHV and –GGV wa s lower than that for CapG severing mutant and resembled wild type CapG. All three mutants were stil l capable of capping the barbed ends of actin filame nts. However, all three mutants lost the ability to sever filamentous actin. These observations demonstr ate that length and char ge interactions in the domain I-II linker are critical for proper se vering function, and that alterations in length are less detrimental to the capping f unction of the CapG severing mutant.

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1 CHAPTER 1 INTRODUCTION Cell movement is essential for such activities as chemotaxis, wound healing, pathogen invasion, and immune function (M arx, 2003). The actin cytoskeleton and actin-regulatory proteins play vital roles in generating the forces and shape changes required for cell movement. Actin Actin is a globular protein th at is separated into two lobes by a cleft that forms the ATP-binding site (Bettinger et al., 2004). ATP-bound actin monomers (globular or G-actin) can assemble into filaments (filamentous or F-actin), associated with the hydrolysis of ATP. Actin filaments are co mposed of two strands that twist around one another to form a double right-handed helix. These filaments are further organized by accessory proteins into diverse structures th at carry out actin's cytoplasmic functions. There are three isoforms of actin in higher eukaryotes; -actin is muscle specific, whereas and -actin are found in all cell ty pes (Bettinger et al., 2004). Actin assembly from G-actin to F-actin occurs in a three-step process. The first step is also the rate-limiting step (called nucl eation or the lag phase). At least three actin monomers need to aggregate for nucleation to begin. After the initial setup, the growth or elongation phase occurs as subunits rapidly add to the nucleated filament ends. The final step is the steady-state phase, when a balance exists between the rate of new subunits adding to the filament ends and the rate of subunits leaving the ends. A value known as the critical concentr ation designates the concentra tion of free subunits left in

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2 solution once the steady state is reached. Be low this value actin will depolymerize and above the critical concentration it will polymerize (Oosawa and Asakura, 1975). Once assembled into a filament, actin has two distinct ends: a barbed end and a pointed end. The pointed end is known hist orically for the arrow-like appearance of myosin heads bound along the fila ment (Alberts et al., 2002). The barbed end is also called the plus end, and is the more dynamic of the two ends; meaning that monomers will grow and depolymerize faster at this end than at the pointed or minus end. Actin-Regulatory Proteins A myriad of proteins are dedicated to actin regulation. These in clude proteins that nucleate, bind monomers, side bind, bundle/ cross-link, cap and sever actin (Ayscough, 1998). Actin binding-proteins are also found in the nucleus (Bettinger et al., 2004). I focused on the capping and severing functions of actin regulatory proteins. When one end of the actin filament is capped, no furt her polymerization or depolymerization occurs at that end. A protein that severs binds alongside the actin filament and inserts itself between adjacent subunits to weaken the noncovalent bonds of the filament, until it severs and binds to one of the ba rbed ends (Ferrary et al., 1999). Both capping and severing abilities play essential roles in cell motility. When a protein severs an actin filament, new ends are created as old filaments are dismantled. Consider a macrophage in fierce pursuit of a bacterium. Inside the macrophage is an actin network that is conti nuously chopped up by severing prot eins and redistributed to form new positions that the macrophage must assume to catch its bacterial prey. A model that illustrates the importance of ca pping is cellular necrosis. When a cell dies, both G-actin and F-actin pour into the extracellular space. Conditions in this space (ionic strength, pH, and temper ature, etc.) are conducive fo r rapid polymerization of the

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3 actin monomer pool which leads to increased viscosity and potential damage to the surroundings. Proteins that cap actin stop this elongation, reduce viscosity, and thwart uncontrolled polymerization (Burtnick et al., 1997). Gelsolin, an 82 kDa protein that contains six domains and has the ability to cap and sever actin filaments, was discovered in 1979 by Yin and Stossel. Gelsolin along with related proteins including villin, severin, adseverin, fragmin and CapG, constitute the gelsolin superfamily. Origins of the Name CapG My study focused on CapG. This gelsolin family member was discovered in 1986 by Southwick and DiNubile. CapG has al so been called macrophage capping protein (MCP), gCap39, and Myc basic motif homolog-I (Mbh1). Then in 1994 under a suggestion from the Genome Data Base organi zers at Johns Hopkins University Medical School, researchers agreed on the name CapG. The “Cap” denotes the protein’s ability to cap the barbed ends of actin filaments, but not to sever them. The “G” signifies that the derived amino acid sequence most closely rese mbles gelsolin (Mishra et al., 1994). Due to this relatedness, gelsolin will be compared alongside CapG for most of the discussion. Characterizing CapG CapG has a molecular weight of 38 kDa. There are three 14 kDa repeat subdomains, as compared to gelsolin which c ontains six of these 14 kDa repeats (Mishra et al., 1994). CapG has a stokes radius of 3.0 nM, and its isoelectric point is 6.6, migrating as a single polypeptide (Southwick and DiNubile, 1986). According to Mishra et al. (1994) the CapG gene is 16.6 kb with 10 exons and 9 introns. The open reading frame is 6.9 kb with 9 exons, 8 introns, and 3 splice sites that

PAGE 15

4 are nearly identical to the human gelsolin gene. The proximal short arm of chromosome 2 houses the CapG gene. Evolution/Sequence Comparison In 1994 Mishra et al. asked whether gelsolin and/or villin may have arisen from CapG gene duplication. However the pathway to this evolutionary jigsaw appears to be more complex than simple CapG gene duplica tion. One clue to the complexity of this issue is derived from sequence comparisons. When CapG is compared to other gelsolin family proteins, the sequence identity vari es depending on the region of the protein as well as its origin (i.e., mammalian vs. othe r). The first three domains of mammalian gelsolin show a 49% sequence identity to Ca pG. However, the last three domains are only 16% identical, thus casting doubt on the possibility of CapG gene duplication (Dabiri et al., 1992). Mammalian villin also showed this sequence identity discrepancy with a 41% identity in the fi rst three domains, and only a 10% identity in the last three domains (Dabiri et al., 1992). Other family members with three domains include fragmin ( Physarum polycephalum ) and severin ( Dictyostelium discoideum ), that show sequence identity to CapG of 30% and 25%, re spectively (Dabiri et al., 1992). CapG Sequence, Structure and Function CapG possesses three domains. Accordi ng to Yu et al. (1991) domain I is gelsolin’s equivalent for actin monomer binding. CapG domain I is dependent on Ca2+ for actin binding where as gelsolin does not share this dependency. Gelsolin has 6 domains, referred to as G1-G6. In gelsol in’s N-terminal half there is an actin side-binding site (domains II and III), and a monomer binding site (domain I), but domain I and half of domain II are required for se vering actin filaments (Yu et al., 1991).

PAGE 16

5 In 1991 Prendergast and Ziff probed for candi date factors to interact with the c-Myc oncoprotein. They found CapG and therein a distantly related basic/helix-loop-helix (B/HLH) DNA-binding mo tif. The real catch was the potential nuclear localization signal dubbing CapG a nuclear protein. In 1993 this fact was solidified by Onoda, Yu and Yin who discove red that CapG was i ndeed both a nuclear and cytoplasmic protein. Another 1993 pa per by Onoda and Yin showed that CapG could be phosphorylated and th at this phosphorylated form (e nhanced by okadaic acid) of CapG was abundant in the nucleus, perhaps prov iding some sort of s ubcellular regulation. CapG’s size of 38 kDa places it on the borde r line of being actively transported or passively diffused into the nucleus which b ecomes hampered and inefficient as protein size approaches 20-40 kDa (Van Impe et al., 2003). They also reported that CapG did not possess the nuclear localizat ion signal typified by other nu clear actin-binding proteins (i.e., supervillin and zyxin). Severin and fr agmin were found to contain Rev-like nuclear export signals in their N-terminus which cont rolled their passage from the nucleus to cytoplasm. In terms of function, CapG has the ability to cap the barbed ends of actin filaments. It should again be noted that CapG unlike its other family members is not capable of severing actin filaments. Regulation of CapG Capping activity in CapG requires mi cromolar concentrations of Ca2+. CapG can dissociate from actin filament ends readily either by a decrease in Ca2+ levels to submicromolar concentrations (Southwic k and DiNubile, 1986) or by increasing phosphoinositide 4,5-bisphosphate concentrations (Yu et al., 1990). Gelsolin uncapping

PAGE 17

6 requires both increases in phosphoino sitides and a decrease in Ca2+ concentrations (Yu et al., 1990). Cellular Distribution With the exception of platelets, CapG is found in nearly all ce lls usually at low concentrations of approximately 0.05% of the to tal protein. However, CapG is one of the most abundant cytoplasmic proteins in macrophages and dendritic cells, where it accounts for 0.9-1% of the total cytoplasmic protein (Dabiri et al., 1992; Parikh et al., 2003). Yu et al. (1990) also pur ified CapG from human plasma where it appeared to be a minor component. Gelsolin also has a broad tissue distribution and is found in platelets as well as smooth and skeletal musc le cells (Dabiri et al., 1992). Membrane Ruffling Loss of CapG affects cell movement (Witke et al., 2001). Macrophages from CapG knockout mice demonstrate decreases in phagocytosis and membrane ruffling. In 2003, Parikh et al. once again studied Ca pG knockout mice with their impaired membrane ruffling and found the null mice to be more susceptible to infection by the food borne pathogen Listeria monocytogenes but demonstrated normal susceptibility to Salmonella enterica Serovar Typhimurium infection. This suggested that patients with unexplained listeriosis may be suffering from a CapG deficiency, although no current CapG defects in humans have been described (Parikh et al., 2003). Mutations to CapG In 1995 Southwick reported a series of ga in-of-function mutations to CapG which rendered the protein capable of severing actin filaments. First the sequences of CapG, gelsolin, villin, severin and fragmin were sc rutinized for consensus sequences likely to confer severing function (Figure 1-1). Two CapG sequences we re found to be divergent.

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7 Thus mutations to convert the divergent stretches to gelso lin sequences were performed and a gain-of-function resulted. The resultant mutant CapG 124GFKHV128 and 84LDDYLGG90 still did not sever as we ll as gelsolin (gelsolin required one-fiftieth the concentration to cause severing comparable to the 124GFKHV128 and 84LDDYLGG90 mutant). The LDDYLGG region is known to be the central region of a long -helix that interacts with the subdomain 1 and 3 cl eft of the actin monomer (Southwick, 1995). Further mutations were performed to CapG 124GFKHV128 and 84LDDYLGG90 again as dictated by sequence divergence in othe r gelsolin family members. Amino acids 129-137 of CapG were made identical to gelsolin. The new mutant CapG 124GFKHVVPNEVVVQR137 and 84LDDYLGG90 proved to be a more potent severer than the original gain-of-function by 3-4 fold (Zhang et al., unpublished). Severing Mutant Mutations My contribution to understanding how CapG ’s structure relates to its kinetic functions was to perform truncation and additi on mutations to the already mutated CapG 124GFKHVVPNEVVVQR137 and 84LDDYLGG90 (here called CapG severing mutant). One addition and two deletions were made in sets of three amino acid changes to the linker region between domains I and II that is believed to hold spatial significance to the severing ability previously bestowed upon this mutant. It was determined that three amino acid changes would be sufficient to alter this linker’s length, and might be expected to alter CapG severing mutant’s function as reflecte d by kinetic analysis experiments.

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8 Mutations performed in my studies were as follows: CapG severing mutant 15KYQEGGVESGFKHVV Deletion of –KHV 115KYQEGGVESGF V Addition of +AAA 115KYQEGGVESAAA GFKHVV CapG severing mutant 115KYQEGGVESGFKHVV Deletion of –GGV 115KYQE ESGFKHVV I postulated that these mutations would not alter the ability of CapG severing mutant to cap the barbed ends of actin filaments. However, given the complexity of the severing mechanism, I proposed that the mutatio ns would modify the proteins ability to sever actin filaments. Figure 1-1. Sequence comparisons of CapG, ge lsolin, villin, fragmin, and severin. A) amino acid sequences were compared w ithin the region previously suggested to be important for severing. The shaded areas show regions of high identity. Two regions (in B and C) revealed devi ation of the CapG sequence (boldface letter in unshaded blocks from the c onsensus sequences shared by the severing proteins. Gaps are designated by dash es. The region of identity to the and -actin sequence in underlined in B. The region highly conserved among the severing proteins, but different in CapG, is underlined in C. (Southwick, F.S. 1995. Gain-of-function mutations conferri ng actin-severing ac tivity to human macrophage cap G. J Biol Chem 270:45-8. Figure 1, page 46.)

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9 CHAPTER 2 MATERIALS AND METHODS Site-Directed Mutagenesis Human CapG cDNA was cloned into pET12a at NdeI and SalI polylinker sites as previously described by Dabiri et al. (1992). Mutants were generated by Stratagene’s QuickChange Site-directed Mutagenesis Kit (S tratagene, La Jolla, CA) according to the manufacturer’s instru ctions. PCR melting, annealing, a nd elongation temperatures used were 95, 55, and 68 C respectively. Protein Expression and Purification Clones in BL21(DE3) were grown in 1L LB at 37 C containing 50 g/ml carbenicillin to an A600 = 0.6 and induced with 0.5 mM isopropyl-1-thio-D-galactoside (IPTG). Upon induction CapG severing muta nts –KHV and –GGV were grown for 3 h at 25 C. CapG severing mutant +AAA remained at 37 C for 3 h post-induction. To harvest cells, centrifugation wa s carried out at 5,000 X g for 5 min at 4 C. Pellets were resuspended in 10 ml lysis buffer (10 mM Imidazole, 1 mM EGTA, 1 mM DTT, 1X mini cocktail protease inhibitor tablet from Roch e Applied Science, Mannheim, Germany) and then freeze thawed three times to lyse cells. 100 g/ml lysozyme and 10 g/ml DNaseI was added and samples shaken for 30 min at 25 C before sonication at 80% power for 3 min. The cell lysates we re then centrifuged at 40,000 X g for 30 min at 4 C and resultant supernatant and pellet fractions were analyzed on Coomassie Blue stained SDS-polyacrylamide gels.

PAGE 21

10 The supernatants were loaded onto DEAE ion-exchange columns (Amersham Biosciences). If necessary samples were c oncentrated in a 9 kD Apollo 20 ml high performance centrifugal spin concentrators (Orbital Biosciences, LLC, Topsfield, MA) for 15 min at 170,000 X g 4 C, and repeated until a volume of 5 ml was attained before being loaded onto a HiPrep desalting column (Amersham Biosciences). Coomassie Blue stained SDS-polyacrylamid e gels were employed to analyze the proteins purity. Pure fracti ons were pooled and stored in 30% ethylene glycol. The method of Bradford was used to obtain pr otein concentrations using BSA standards (Bradford, 1976). CapG severing mutant was grown and harves ted following the same protocol as the mutants except that CapG se vering mutant was grown at 37 C for 2 h post-induction. Actin Purification Actin was purified from skeletal muscle (rabbit, porcine, chicken) and conjugated to N -(1-pyrenyl) iodoacetamide at actin’s Cys 374 as previously described (Young et al., 1990). Briefly, actin was made into an acetone powder (Spudich and Watt, 1971; Pardee and Spudich, 1982) by mincing muscle and extracting it with 1L 0.1 M KCl, 0.15 M potassium phosphate, pH 6.5 before being filt ered through cheesecloth, stirred in 2L 0.05 M NaHCO3 and then cheesecloth filtered again. This extraction was followed by the addition of 1L 1 mM EDTA, pH 7.0 and then two extractions of 2L of distilled H2O. The last five extractions were performed with 1L acetone. The filtered residue was air dried in glass evaporating dishes overnight in a fume hood. The resulting acetone powder was extracted in Buffer-G (5 mM Tris, 0.1 mM CaCl2, 0.2 mM ATP, 0.2 mM DTT, 0.01% Sodium Azide, pH 8.0) and filtered through chee se cloth before being ultracentrifuged for 40 min at 4 C, 28,000 X g The supernatant was slowly st irred at room temperature for

PAGE 22

11 1 h while adding 2 l/ml MgCl2 and 52 mg/ml KCl to polymer ize the actin, and followed by centrifugation for 3 h at 4 C, 49,300 X g The actin pellet was resuspended in a minimal volume of Buffer-G and dialyzed against fresh Buffer-G for 3 days to depolymerize the filaments. Residual Factin was removed by ultracentrifugation for 45 min at 4 C, 49,300 X g The supernatant was loaded onto a S-200 Column equilibrated with Buffer-G and eluted with Buffer-G. Purified monomeric actin was labeled with pyrene, N -(1-pyrenyl) iodoacetamide, accord ing to standard procedures (Kouyama and Mihashi, 1981). The critical c oncentration was determined before each assay and found to be between 0.01 M and 0.08 M. (Cooper et al., 1983). Kinetic Assays Subcritical Actin Monomer Fluorescence Assay Increasing amounts of protein were added in a glass cuvette with monomeric actin just below the critical concentration at 300 nM G-Actin in S1 buffer (10 mM imidazole, 100 mM KCl, 0.4 mM MgCl2, 0.1 mM CaCl2, 0.1 mM ATP, 0.5 mM DTT, pH 7.6). The solution was mixed 5X after addition of protei n to ensure the formation of protein-actin complexes. These complexes are weakly fl uorescent due to the conformation change of the actin molecule labeled with pyrene. Fluorescent intensity of these protein-actin complexes was measured and once the intens ity values stabilized, additional CapG mutant protein was added to the solution, mixed 5X, and the intensity was measured again. The final intensity values were plotted against protein concentration. Fluorescence was monitored on a HORIBA Jobin Yvon FluoroLog 3 spectrofluorometer (Edison, NJ) with ex = 366 nm and em = 385 nm while maintaining a slit length at 5.0 mm. Data was collected us ing DataMax Software (HORIBA Jobin Yvon Edison, NJ).

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12 Monomer Sequestration Assay 2 M Actin was polymerized in S1 buffer (10 mM imidazole, 100 mM KCl, 0.4 mM MgCl2, 0.1 mM CaCl2, 0.1 mM ATP, 0.5 mM DTT, pH 7.6) using gelsolin capped F-actin nuclei, molecular ratio 1:20 (g elsolin to actin, final concentration of F-actin seeds of 0.1 M). Assembly rates of the point ed end were measured in the presence of increasing concen trations of capping protein, 3 M G-actin, and 200 l 2XP buffer (20 mM imidazole, 0.2 mM KCl, 4 mM MgCl2, 2 mM ATP, 2 mM DTT, pH 7.4). The capping protein sequestered monomers aw ay from the G-actin pool resulting in a decrease in fluorescence of pointed end filament assembl y. Fluorescent intensity was observed with respect to time on the FluoroLog 3 (same settings as described before). Capping Assay 2 M Actin was allowed to polymerize ove rnight in S1 buffer (10 mM imidazole, 100 mM KCl, 0.4 mM MgCl2, 0.1 mM CaCl2, 0.1 mM ATP, 0.5 mM DTT, pH 7.6) at 25 C in the dark. F-Actin was diluted 1:20 belo w the critical concentration in S1 buffer to 100 nM in a glass cuvette. F-Actin was mechanically sheared 5X with an extended length p200 pipette tip (Fisher Scientific, Suwanee, GA) to create new barbed and pointed ends that rapidly de polymerized as shown by a decr ease in fluorescent intensity. Increasing amounts of capping protein were a dded to the buffer which capped the barbed ends of the actin filaments and retained fluorescent intensit y. Fluorescence was monitored over time on the FluoroLog 3 (s pectrofluorometer settings as described before). Severing Assay 2 M Actin was allowed to polymerize in S1 buffer (10 mM imidazole, 100 mM KCl, 0.4 mM MgCl2, 0.1 mM CaCl2, 0.1 mM ATP, 0.5 mM DTT, pH 7.6) using gelsolin

PAGE 24

13 capped actin filament molar ra tio 1:200 (gelsolin to actin). F-Actin was diluted 1:20 below the critical concentrati on in S1 in buffer to 100 nM in a glass cuvette and mixed gently 5X to avoid shearing filaments (and k eep fluorescence at a steady intensity) before adding capping protein while mixing another 5X. Severing proteins create new barbed and pointed ends that depolymerize belo w the critical concentration resulting in decreased fluorescent intensity. Fluorescen ce was observed with respect to time on the FluoroLog 3. Spectrofluorometer settings were the same. Table 2-1. Primer design for CapG severing mutants. +AAA—foward 119 120 121 122 123 124 125 126 127 128 129 119 120 121 122 123 124 125 126 127 128 129 130 131 132 GGT GGT GTG GAG TCA GCG GCG GCG GGA TTT AAA CAC GTG GTT G G V E S A A A G F K H V V Gly Gly Val Glu Ser Ala Ala Ala Gly Phe Lys His Val Val +AAA—reverse AAC CAC GTG TTT AAA TCC CGC CGC CGC TGA CTC CAC ACC ACC -KHV—forward (d elete K126 H127 V128) 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 GGT GGT GTG GAG TCA GGA TTT AAA CAC GTG GTT CCG AAC GAA GTT G G V E S G F K H V V P N E V Gly Gly Val Glu Ser Gly Phe Ly s His Val Val Pro Asn Glu Val -KHV—reverse AAC TTC GTT CGG AAC CAC GTG TTT AAA TCC TGA CTC CAC ACC ACC -GGV—forward (delete G119 G120 V121) 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 CGG GGC CTC AAG TAC CAG GAA GGT GGT GTG GAG TCA GGA TTT AAA CAC R G L K Y Q E G G V E S G F K H Arg Gly Leu Lys Tyr Gln Glu Gly Gly Val Glu Ser Gly Phe Lys His -GGV—reverse GTG TTT AAA TCC TGA CTC TTC CTG GTA CTT GAG GCC CCG

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14 CHAPTER 3 RESULTS Purification of CapG Mutants All mutants were purified as outlined in Materials and Methods. CapG severing mutant +AAA was first passed through a DE AE column and analyzed on Coomassie Blue stained SDS-polyacrylamide gels. Fracti ons 42 to 61 were concentrated in a 9 kD Apollo 20ml high performance centrifugal spin concentrator before being loaded onto a large HiPrep desalting column. The protein was found to be of 99% and 96% purity and collected from fractions 5-7 and 10-11, re spectively (Figure 3-1A ). CapG severing mutant –GGV was found to be of 99% and 90% purity and collected from fractions 17-19 and 20-24, respectively, of a DEAE column (Figure 31B). CapG severing mutant –KHV was collected from pooled fractions 32 -37 from a DEAE column and found to be of 98% purity (Figure 3-1C). Structure-Function Analysis Effects of Actin Monomer Binding by CapG Mutants We first assessed the ability of the mutant protein to bind monomeric actin. A subcritical actin monomer fluorescence a ssay (which operates based on change in fluorescence intensity of a subcritical concentr ation of pyrenyl actin ; refer to Materials and Methods), was performed on all mutant a nd wild type proteins The conformational change in actin upon polymerization is r eadily detected by the sulfhydryl reagent N -(1-pyrenyl) iodoacetamide. This probe was found to display a fluorescence spectrum more sensitive than previously reported pr obes, and has since been the standard for

PAGE 26

15 kinetic assessment (Kouyama and Mihashi, 1981). When pyrenyl actin undergoes polymerization a 20 fold increase in fluor escence is observed (Kouyama and Mihashi, 1981). However, in the subcritical monomer assay where no polymerization occurs, only a 2 to 3 fold increase in fluorescence was see n. This small rise in fluorescence reflects a conformational change in monomeric actin as capping protein-actin complexes are formed (Southwick and DiNubile, 1986; Young et al., 1990 and 1994). In Figure 3-2B CapG severing mutant +AAA reaches peak fluorescence at 2000 nM. The KD (dissociation) was determined to be 1000 nM. CapG severing mutant –GGV achieved saturation at 5000 nM (Figure 3-2C) yielding a KD of 2,500 nM. CapG severing mutant has a higher affinity for actin monomers with a KD of 150 nM (Figure 3-2A), than the +AAA and –GGV mutants. Wild type CapG has a KD reported to be 1000 nM (Young et al., 1990). For reasons that are unknown, and in cont rast to the other mutants, the –KHV mutant was not amenable to study using th e subcritical actin monomer fluorescence assay. To overcome this I employed a second assay known as the monomer sequestration assay to determine the monomer binding affi nity in this mutant. For this monomer sequestration assay, gelsolin is incubated with actin (1:20) which caps the barbed end of the actin filament leaving only the pointed en ds open for elongation. G-actin is added to the reaction cuvette above th e critical concentration of the pointed end allowing polymerization at that end. When –KHV mutant is added it cannot cap the pointed end, therefore any reduction in the ra te of pointed end assembly would result from its ability to bind and prevent actin monomers from addi ng to the filament (often termed monomer sequestration). Figure 3-2D shows that very hi gh amounts of CapG severing mutant

PAGE 27

16 –KHV are required to inhibit actin assembly. 3000 nM is required to completely sequester actin monomers and the KD is estimated to be 1,500 nM. The affinity of this mutant for actin monomers is markedly reduc ed when compared to the primary severing mutant (Figure 3-2A). All mutants, + AAA, –GGV and –KHV displayed this weaker binding affinity with a KD of 1,000 nM, 2,500 nM and 1,500 nM respectively. Capping The ability of the mutants to cap barbed ends of actin filaments was assessed via a capping assay. In this assay, actin was dilu ted below the critical concentration and mechanically sheared as described in Material s and Methods to create free barbed ends that rapidly depolymerize. Pointed ends ar e also created, however, the dynamics at the pointed end are considered negligible in co mparison to the more dynamic barbed end. Capping protein added in increasing amounts to the dilute actin will cap the barbed ends and retain fluorescence as depolymerization halts. In Figure 3-3A CapG severing mutant + AAA begins to slow depolymerization at 2.5 nM. The addition of 10 nM, 20 nM and 100 nM shows similar retention of fluorescence as the +AAA mutant caps the barb ed ends. Note the saturation value of capping protein will never be a perfectly stra ight line due to the slight depolymerization occurring at the pointed end. CapG severi ng mutant –GGV began capping monomers at 0.5 nM (Figure 3-3B). Good fluorescence reten tion is observable at concentrations of 12 nM. CapG severing mutant +AAA and –GGV proved to be most similar to wild type CapG with half maximal capping values of 5 nM and 3 nM respectively. Wild type CapG is reported by Southwick (1995) to have a half maximal capping value of 0.5 nM. CapG severing mutant also has a half maxima l capping value of 0.5 nM (Zhang et al.,

PAGE 28

17 unpublished). CapG severing mutant –KHV was found to have a much higher half maximal capping value between 20 to 50 nM (Fi gure 3-3C) perhaps due to steric charges (reviewed in Discussion section). Severing In order to ascertain whether CapG severi ng mutant’s ability to sever was affected by altering the length of the domain I-II linker, a severing assay was performed (refer to Materials and Methods for a detailed descrip tion). Gelsolin seeded actin was diluted below the critical concentration with care take n in mixing to avoid mechanically shearing the F-Actin (fluorescence will stay constant). When the severing protein is added to the reaction cuvette the filaments will sever creat ing new barbed and pointed ends that will depolymerize resulting in a decrease in fluorescence. Figure 3-4A, B, and C reveal that all CapG severing mu tants +AAA, –GGV and –KHV respectively, did not exhibit any severing activity as observed by the lack of fast depolymerization despite addition of high am ounts of protein. Figure control, CapG severing mutant, displays th is rapid depolymerization. Figure 3-1. Protein gels of th e CapG severing mutant and its triplicate mutants. A) +AAA. B) –GGV. C) – KHV. Coomassie Blue st ained SDS-polyacrylamide gels showing purified protein migrating at 38 kDa. M is protein marker, L is the column load, and numbers denote protein fractions. A B C

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18 180000 190000 200000 210000 220000 230000 240000Relative Fluorescence0246Concentration, M +AAA 40 50 60 70 80 90 100 110Relative Fluorescence0246Concentration, M -GGV 200 250 300 350 400 450 500Relative Fluorescence060120180240300360Time, s 1700nM -KHV 3000nM -KHV 1500nM -KHV 700nM -KHV ControlFigure 3-2. Monomer Binding of CapG Severing Mutant and its triplic ate mutants. A) CapG Severing Mutant. B) +AAA. C) –GGV. Increasing amounts of capping protein were added to 300 nM G-actin in S1 buffer and mixed, and the observed fluorescence increase plotted versus the final concentrations of capping protein. D) Pointed-end growth rate inhibited by the presence of increasing amounts of CapG severing mutant –KHV. Fluorescence was monitored after the addition of 100 nM gelsolin seeded actin added to S1 buffer, capping protein, 3 M G-actin, a nd 2XP buffer (refer to Materials and Methods). Open circles denotes all components mentioned above except for capping protein. 190 200 210 220 230 240Relative Fluorescence0100200300400Concentration, nM mCapG CapG severing mutant C D B A

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19 500 600 700 800 900 1000Relative Fluorescence060120180Time, s 100nM +AAA 2.5nM +AAA 5nM +AAA 10nM +AAA 20nM +AAA C ontro l 500 600 700 800 900 1000Relative Fluorescence 060120180Time, s 50nM -KHV 100nM -KHV 10nM -KHV 20nM -KHV Control 1000 1200 1400 1600 1800 2000 2200 2400Relative Fluorescence060120180Time, s 20nM -GGV 0.5nM -GGV 3nM -GGV 12nM -GGV Control Figure 3-3. Capping activities of the CapG Se vering Mutant triplicate mutants. A) +AAA. B) –GGV. C) – KHV. Depolymerization of the barbed end of 2 M F-actin ( Materials and Methods) dilute d below its critical concentration to 100 nM in S1 buffer is halted by th e addition of increasing amounts of capping protein (filled symbol s) as evidenced by the rete ntion of fluorescence. Note the saturation value of capping prot ein will never be a perfectly straight line due to the slight depolymerization occurring at the pointed end. Open circles denote the disassembly rate of 100 nM pyrene actin in S1 buffer. B C A

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20 600 650 700 750 800 850Relative Fluorescence0204060Time, s 200nM CapG severing mutant 50nM +AAA 100nM +AAA 200nM +AAA Control 650 675 700 725 750Relative Fluorescence0204060Time, s Control 200nM CapG severing mutant 50nM -KHV 75nM -KHV 200nM -KHV 0 200 400 600 800 1000Relative Fluorescence0204060Time, s 6000nM -GGV 5000nM -GGV 10nM -GGV 200nM CapG severing mutant Control Figure 3-4. CapG Severing Mutant triplicate mutants lack severing activity. A) +AAA. B) –GGV. C) –KHV. Increasing am ounts of capping protein was added to gently mixed 2 M F-actin (refer to Materials and Methods) diluted below its critical concentration to 100 nM in S1 buffer. The depolymerization as observed in the CapG severing mutant control (filled circles) denotes severing. Open circles are the same concentration of pyrene actin in S1 buffer. No acceleration in the depolym erization rate was observed in mutants indicating a lack of severing activity. B C A

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21 CHAPTER 4 DISCUSSION Role of the S1-S2 Linker Length on Function My studies were designed to explore the e ffects of length change of the domain I-II linker region of the CapG severing mutant, and are based on the crystal structures of the CapG severing mutant (Figure 4-1) and gelsolin (Burtnic k et al., 2004; Zhang et al., unpublished). The linker region between doma ins I and II in both activated CapG severing mutant and gelsolin is extended at 36 and 30 respectively (Burtnick et al., 2004; Zhang et al., unpublished). This linker positioning is imperative to allow the LDDYL loop steric interactions resulting in se vering of the actin filament (McLaghlin et al., 1993; Zhang et al., unpublished). Structural Determinants of Capping and Severing The model for CapG capping is thought to be similar to gelsolin G1-G3 capping, which has been made possible through the efforts of X-ray crystallography, electron microscopy (EM), and nuclear magnetic resonance (NMR) spectroscopy (McGough et al., 2003). Figure 4-2 depicts G1 bound to subunit 1 at the very end of the actin filament and G2 bound in an area bridging subunit 1 an d 3 (McGough et al., 2003). Past studies by Irobi and colleagues place G1 and G2 on adjacent monomers of the same long-pitch helix of the filament. In eith er case, the triplicate mutant s of the CapG severing mutant did not disrupt capping activity, indicating that larger length alterations in the domain I-II linker region may be needed to misalign domain I and impair capping function.

PAGE 33

22 Severing is a more complex function. In full length gelsolin, severing is initiated by Ca2+ which causes the protein to undergo ma ny conformational changes (Burtnick et al., 2004). For instance Sun, et al. (1999) reports that in the presence of Ca2+ the extended -sheet between G4 and G6 is broken al ong its interface, G6 swings out from G4 to form new contacts with G5, and actin becomes situated into the unoccupied G6 space to create an intermolecular Ca2+ binding site coordinated by G4 and actin. Ultimately for severing, G2-G3 attaches alongside an actin filament while the flexible G1-G2 linker extends between two adjacent pr otomers on the long-pitched actin strand (Burtnick et al., 2004). Simultaneously, in a separate direction the G3-G4 linker and G6 wrap over the filament’s surface and direct G1 and G4 to their binding sites (Burtnick et al., 2004). A concerted pincer mo tion of G1 and G4 imparts a st eric strain so great that enough non-covalent bonds between the actin monomers in the filament below are weakened and the filament is severed (Burtn ick et al. 2004; Sun et al., 1999). Figure 4-3 illustrates the severing process in gelsolin. CapG and other members of the gelsolin superfamily are thought to possess the same mechanism of attachment to the actin molecule as the gelsolin domain I and II linking peptide, based on sequence homology (Irobi et al., 2003). Although gelsolin severs the filament in two locations, the CapG severing mutant only severs in one location within the actin filament because it can only coordinate three subunits. We predicted that small changes in the domain I-II linker could affect the positioning of the LDDYL severing loop on the actin filament. Th erefore severing would be expected to be more sensitive to alterations in the length of the linker as compared to capping.

PAGE 34

23 Comparison between CapG Severing Mutant and New Mutants To interact with actin, CapG severing mutant must first bind to one or more actin monomers within a filament. Therefore, monomer binding was first assessed. Deleting or adding amino acids in triplicate was detr imental to CapG severing mutants’ monomer binding affinity as evidenced by a 7-fold reduction in +AAA, a 17-fold reduction in –GGV and a 10-fold reduction in –KHV. When compared to wild type CapG with a KD reported to be 1000 nM (Young et al., 1990) the triplicate mutants’ affinity for monomeric actin is comparable (a KD of 1000 nM, 2,500 nM and 1,500 nM for +AAA, –GGV and –KHV mutants re spectively, Table 4-1). In Zhang et al., unpublished, it has been postulated that CapG severing mutant contains two actin binding sites. The addi tion mutant +AAA appears to also possess this second actin binding site as evidenced by the decrease in fluorescence at very high concentrations of the mutant protein. Th e deletion mutants –GGV and –KHV appear to have a stoichiometry similar to wild type Ca pG with only one actin binding site, since the fluorescence remains elevated at very high concentrations of protein. The ability to cap was also hindered for the –KHV mutant who’s half maximal capping constant was between 20-50 nM as compared to wild type CapG and CapG severing mutant, both with a half maximum capping at 0.5 nM. CapG severing mutant –KHV appeared to be the odd mutant out wh en compared to mutants +AAA and –GGV. This difference could be the consequence of electrostatic charges. The lysine (K), histidine (H), and va line (V) are associated with basic, basic, and neutral charges respectively. Whereas the ot her mutants addition of thr ee neutral alanines (A) and deletion of neutral glycines (G) and valine, would have no effect on charge but simply affect the length of the linker region.

PAGE 35

24 The most profound functional effect was on severing. Figure 4-1 helps illustrate how alterations in length will affect what am ino acid charges get placed adjacent to each other. In mutation site II where the triplica te mutations were made, the side chains are visible. These side chains can attract or re pel the actin residues that make contact with the CapG severing mutant during binding. Side binding of the actin filament is necessary for severing to occur, thus the contacts between the side-binding protein and actin need to be favorable. All triplicate mutants of th e CapG severing mutant did not sever, once again stressing the importance of both lengt h and charge of the linker region between domains I-II in order for severing to occur. Table 4-1. Functional activitie s of CapG and its mutants Protein KD of G-actin (nM) max capping (nM) Severing activity CapG 1000 (Young et al., 1990) 0.5 (Southwick, 1995) No CapG severing mutant 150 0.5 (Zhang et al., unpublished) Yes* CapG severing mutant +AAA 1000 5 No CapG severing mutant -GGV 2,500 3 No CapG severing mutant -KHV 1,500 20-50 No *Half-maximal severing reported to be 30-50 nM (Zhang et al., unpublished).

PAGE 36

25 Figure 4-1. Structure of the CapG severing mutant (Zhang, Y., S.M. Vorobiev, B.G. Gibson, B. Hao, G. Sidhu, V.S. Mishra, E.G. Yarmola, M.R. Bubb, S.C. Almo, and F.S. Southwick. Unpublished. A CapG gain-of-function mutant reveals critical structural and functio nal determinants for actin filament severing.)

PAGE 37

26 Figure 4-2. Possible model of filament capping by gelsolin. Actin filaments are represented by space-filling models orie nted with the minus or slow-growing end up. Actin subunits from one long-pitch strand are colored yellow and those from the other are colored gray (McGough, A.M., and C.J. Staiger, J.K. Min, and K.D. Simonetti. 2003. The gelsolin family of actin regulatory proteins: modular structures, versat ile functions. FEBS Lett. 552:75-81. Figure 2A, page 78.) G1=blue violet G2=pink G3=green G4=aqua G5=red G6=lime green

PAGE 38

27 Figure 4-3. Sequence of events during severing of actin by fully activated gelsolin. Actin protomers shown in blue. Gelso lin subunits are multi-colored ovals. Reprinted by permission from Macmillan Publishers Ltd: [The EMBO Journal] (Burtnick, L.D., D. Urosev, E. Ir obi, K. Narayan, and R.C. Robinson. 2004. Structure of the N-terminal half of gelsolin bound to actin: roles in severing, apoptosis and FAF. EMBO J 23:2713-22. Figure 2B, page 2716.), copyright (2004)

PAGE 39

28 CHAPTER 5 CONCLUSION The reorganization of the actin network by ma ny different proteins is essential for cell movement. The role of CapG in the cell is to cap the barbed ends of actin filaments thereby preventing further monomer growth from that end. CapG is unique among its gelsolin family members in that it does not possess the ability to se ver filamentous actin. Severing is necessary for cellular plasticity; it er ases old actin networks so that new ones can be formed (Ferrary et al., 1999). CapG was mutated to resemble gelsolin in two separate locations, 124GFKHVVPNEVVVQR137 and 84LDDYLGG90 to create the CapG severing mutant (Zhang et al., unpublished). When the length of the region linking domains I-II is altered by three amino acids, severing function is lost but capping activity is preserved. To further investigate how lengt h translates structurally one would need to perform a crystallographic analysis to view the exact positioning of the affected domain I-II linker in the triplicate mutants comp ared to the CapG severing mutant.

PAGE 40

29 LIST OF REFERENCES Alberts, B., A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. The Cytoskeleton in Molecular biology of the cell, 4th ed p.916, Garland Science: New York, New York, 2002. Ayscough, K.R. In vivo functions of actin-binding proteins. 1998. Curr Opin Cell Biol 10:102-11. Bettinger, B.T., D.M. Gilbert, and D. C. Amburg. 2004. Actin up in the nucleus. Nat Rev Mol Cell Biol 5(5):410-5. Bradford, M.M. 1976. A rapid and sensitive me thod for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-54. Burtnick, L.D., E.K. Koepf, J. Grimes, E.Y. Jones, D.I. Stuart, P.J. McLaughlin, and R.C. Robinson. 1997. The crystal structure of plas ma gelsolin: implic ations for actin severing, capping, and nucleation. Cell 90:661-70. Burtnick, L.D., D. Urosev, E. Irobi, K. Na rayan, and R.C. Robins on. 2004. Structure of the N-terminal half of gels olin bound to actin: roles in severing, apoptosis and FAF. EMBO J 23:2713-22. Cooper, J.A., S.B. Walker, and T.D. Pollar d. 1983. Pyrene actin: do cumentation of the validity of a sensitive assa y for actin polymerization. J Muscle Res Cell Motil 4: 253-62. Dabiri, G.A., C.L. Young, J. Rosenbloom, and F.S. Southwick. 1992. Molecular cloning of human macrophage capping protei n cDNA. A unique member of the gelsolin/villin family expressed primarily in macrophages. J Biol Chem 267:16545-52. Ferrary, E., M. CohenTannoudji, G. PehauArnaudet, A. Lapillonne, R. Athman, T. Ruiz, L. Boulouha, F. El Marjou, A. Doye, J. J. Fontaine, C. Antony, C. Babinet, D. Louvard, F. Jaisser, and S. Robine. 1999. In vivo, villin is required for Ca(2+)dependent F-actin disruption in intestinal brush borders. J Cell Biol 146(4):81930. Irobi, E., L.D. Burtnick, D. Urosev. K. Na rayan, and R.C. Robinson. 2003. From the first to the second domain of gelsolin: a common path on the surface of actin: FEBS Lett 552:86-90.

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30 Kouyama, T. and K. Mihashi. 1981. Fluorimet ry study of N-(1-pyrenyl)iodoacetamidelabelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromysin. Eur J Biochem 114:33-8. Marx, J. 2003. How cells step out. Science 302: 214-6. McGough, A.M., and C.J. Staiger, J.K. Mi n, and K.D. Simonetti. 2003. The gelsolin family of actin regulatory proteins: modular structures, versatile functions. FEBS Lett 552:75-81. Mishra, V.S., and E.P. Henske, D.J. Kwiatkowski, and F.S. Southwick. 1994. The human actin-regulatory protein cap G: gene structure and chromosome location. Genomics 23:560-5. Onoda, K., and H.L. Yin. 1993. gCap39 is phos phorylated. Stimulati on by okadaic acid and preferential association with nuclei. J Biol Chem 268(6):4106-12. Onoda, K., F.X. Yu, and H.L. Yin. 1993. gCa p39 is a nuclear and cytoplasmic protein. Cell Motil Cytoskeleton 26(3):227-38. Oosawa, F., and S. Asakura. 1975. Thermodyna mics of the polymerization of protein. Academic Press New York 41-55, 90-108. Pardee, J.D., and J.A. Spudich. 1982. Purification of muscle actin. Methods Cell Biol 24:271-89. Parikh, S.S., S.A. Litherland, M.J. Clare-Salzle r, W. Li, P.A. Gulig, and F.S. Southwick. 2003. CapG -/mice have specific host defense defects that render them more susceptible than CapG +/+ mice to Listeria monocytogenes infection but not to Salmonella enterica serovar Typhimurium infection. Infect Immun 71(11):658290. Prendergast, G.C., and E.B. Ziff. 1991. Mbh1: a novel gelsolin/severin-related protein which binds actin in vitro and exhibits nuclear localization in vivo EMBO J 10:757-66. Southwick, F.S. 1995. Gain-of-function mutatio ns conferring actin-s evering activity to human macrophage cap G. J Biol Chem 270:45-8. Southwick, F.S., and M.J. DiNubile. 1986. Rabbit alveolar macrophages contain a Ca2+sensitive, 41,000-dalton protein which reversibly blocks th e “barbed” ends of actin filaments but does not sever them. J Biol Chem 261:14191-5. Spudich, J.A., and S. Watt. 1971. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolyti c fragments of myosin. J Biol Chem 246:4866-71.

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31 Sun, H.Q., M. Yamamoto, M. Mejillano, a nd H.L. Yin. 1999. Gelsolin, a multifunctional actin regulatory protein. J Biol Chem 274(47):33179-82. Van Impe, K., V. de Corte, L. Eichinger, E. Bruyneel, M. Mareel, J. Vandekerckhove, and J. Gettemans. 2003. The nucleo-cytoplas mic actin-binding protein CapG lacks a nuclear export sequence present in structurally related proteins. J Biol Chem 278:17945-52. Witke, W., W. Li, D.J. Kwiatkowski, and F.S. Southwick. 2001. Comparisons of CapG and gelsolin-null macrophages: demonstr ation of a unique role for CapG in receptor-mediated ruffling, phagocytosis, and vesicle rocketing. J Cell Biol 154:775-84. Yin, H.L., and T.P. Stossel. 1979. Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-depende nt regulatory protein. Nature (London) 281:583-6. Young, C.L., A. Feierstein, and F.S. S outhwick. 1994. Calcium regulation of actin filament capping and monomer bi nding by macrophage capping protein. J Biol Chem 269:13997-4002. Young, C.L., F.S. Southwick, and A. Weber. 1990. Kinetics of the interaction of a 41kilodalton macrophage capping protein with actin: promo tion of nucleation during prolongation of the lag period. Biochemistry 29:2232-40. Yu, F.X., P.A. Johnston, T.C. Sudhof, a nd H.L. Yin. 1990. gCap39, a calcium ionand polyphosphoinositide-regulated actin capping protein. Science 250(4986):1413-5. Yu, F.X., D.M. Zhou, and H.L. Yin. 1991. Chimer ic and truncated gCap39 elucidate the requirements for actin filament severing a nd end capping by the gelsolin family of proteins. J Biol Chem 266(29):19269-75.

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32 BIOGRAPHICAL SKETCH Andrea Roebuck was born in Michigan and m oved to Florida at the age of four. Studies at the University of Florida (UF) be gan in June 1999, with a Bachelor of Science awarded in microbiology and cell science in May 2003. Andrea continued her graduate studies at UF to obtain a Ma ster of Science in molecula r genetics and microbiology in May 2006.


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Title: Kinetic Analysis of Truncation and Elongation Mutants of the CapG Severing Mutant
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
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KINETIC ANALYSIS OF TRUNCATION AND ELONGATION MUTANTS OF THE
CapG SEVERING MUTANT















By

ANDREA ROEBUCK


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Andrea Roebuck

































To my mother, whose presence here today exemplifies perseverance.















ACKNOWLEDGMENTS

I would first like to extend my thanks to my mentor (whose patience is great) and

to my supervisory committee for their thoughtful input. Members of my lab should be

commended for their support. I thank my family for their encouragement and love.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ........._ .. ........ .... .... .... ................. .. .... vii

LIST OF FIGURES ............ ...................... .. ................ viii

LIST OF ABBREVIATIONS ................................................................ .............. ix

A B ST R A C T .......... ..... ...................................................................................... x

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

A c tin ................................................................................. 1
A ctin-R regulatory Proteins .................................................. .............................. 2
O origins of the N am e C apG ................................. ......... .................................. 3
C characterizing C apG ................. ................................ ...... ........ .......... .......
Evolution/Sequence C om parison............................................ ........... ............... 4
CapG Sequence, Structure and Function ....................................... ...............4
R regulation of CapG .................. ................................ ....... .. ........ .. 5
C cellular D distribution ........................................ ....... ... ........ .. ........ .. ..
M em brane Ruffling ....................... ......................... ............. .... .6
M u station s to C ap G .............. ..... .............................. .......... ........ ........ .. ....
Severing M utant M stations ........................................ .......................................7

2 M ATERIALS AND M ETHODS ........................................ ........................... 9

Site-D directed M utagenesis......................................... ................................. 9
Protein Expression and Purification ........................................ ......... ............... 9
A ctin P urification ........ .................................................................... ........ ........ 10
Kinetic Assays .............. ............................................... .........................11
Subcritical Actin M onomer Fluorescence Assay ................................................11
M onom er Sequestration A ssay.................................... ..................................... 12
C apping A ssay .................................................................................. 12
Severing A ssay ......................................... .............................. 12





v









3 R E S U L T S .................................................................................. 14

Purification of C apG M utants......................................................................... ... ... 14
Structure-Function A analysis ..................................................................................... 14
Effects of Actin Monomer Binding by CapG Mutants .....................................14
C a p p in g .......................................................................................................... 1 6
S e v e rin g .................................................................................................... 1 7

4 D ISC U S SIO N ............................................................................... 2 1

Role of the S1-S2 Linker Length on Function...........................................................21
Structural Determinants of Capping and Severing ...............................................21
Comparison between CapG Severing Mutant and New Mutants.............................23

5 C O N C L U SIO N ......... ......................................................................... ........ .. ..... .. 28

L IST O F R E FE R E N C E S ............................................... ........................... ................... 29

B IO G R A PH IC A L SK E TCH ..................................................................... ..................32
















LIST OF TABLES

Table pge

2-1. Primer design for CapG severing mutants...... .. .......................... ............ ... .13

4-1. Functional activities of CapG and its mutants ................ ................................. 24
















LIST OF FIGURES


Figure p

1-1 Sequence analysis of CapG, gelsolin, villin, fragmin, and severin............................8

3-1 Protein gels of the CapG severing mutant and its triplicate mutants .....................17

3-2 Monomer Binding of CapG Severing Mutant and its triplicate mutants .................18

3-3 Capping activities of CapG Severing Mutant triplicate mutants.............................19

3-4 CapG Severing Mutant triplicate mutants lack severing activity...........................20

4-1 Structure of the CapG severing mutant. .............. .. ..... ................. 25

4-2 Possible model of filament capping by gelsolin...........................................26

4-3 Sequence of events during severing of actin by fully activated gelsolin .................27
















LIST OF ABBREVIATIONS


A Angstrom
ATP Adenosine 5'-triphosphate
oC Degree Celsius
cDNA Complementary deoxyribonucleic acid
Cys Cysteine
DNA deoxyribonucleic acid
DEAE diethylaminoethyl
DTT dithiothreitol
EDTA ethylenediaminetetraacetic acid
EGTA [ethylenebis(oxyethylenenitrilo)] tetraacetic acid
g gravity
h hour
IPTG isopropyl-1-thio-/f-D-galactoside
kb kilobase
kDa kilodalton
Xem wavelength of emission
kex wavelength of excitation
L liter
LB Luria-Bertani broth
It micro
m meter
min minute
M molar
n nano
PCR polymerase chain reaction
pH hydrogen ion concentration
pyrene N-(1-pyrenyl) iodoacetamide
SDS sodium dodecyl sulfate
Tris tris(hydroxymethyl)aminomethane















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

KINETIC ANALYSIS OF TRUNCATION AND ELONGATION
MUTANTS OF THE CapG SEVERING MUTANT

By

Andrea Roebuck

May 2006

Chair: Frederick Southwick
Major Department: Medical Sciences-Molecular Genetics and Microbiology

Numerous proteins help regulate the actin cytoskeleton network, one of several

systems that necessitate cellular movement. One such regulator is CapG, whose main

role is to cap the barbed ends of actin filaments, thereby preventing actin monomers from

additional polymerization at the barbed end. Other family members in the gelsolin

superfamily (to which CapG belongs) possess the ability to sever actin filaments. CapG

was mutated in two separate locations based on family-member sequence homology, to

create a mutant that could both sever and cap filamentous actin (the CapG severing

mutant). In this work, site-directed mutagenesis was employed to change the critical

domain I-II linker region of the CapG severing mutant by three amino acids. One

insertion and two deletion mutants were created.

The three mutants (CapG severing mutant +AAA, -KHV, and -GGV) were then

subject to three types of kinetic assessments: monomer binding, capping, and severing of

pyrene-labeled actin. The monomer binding curve of CapG severing mutant +AAA was









most similar to its parent CapG severing mutant. However its affinity for actin

monomers and for mutants -KHV and -GGV was lower than that for CapG severing

mutant and resembled wild type CapG. All three mutants were still capable of capping

the barbed ends of actin filaments. However, all three mutants lost the ability to sever

filamentous actin. These observations demonstrate that length and charge interactions in

the domain I-II linker are critical for proper severing function, and that alterations in

length are less detrimental to the capping function of the CapG severing mutant.














CHAPTER 1
INTRODUCTION

Cell movement is essential for such activities as chemotaxis, wound healing,

pathogen invasion, and immune function (Marx, 2003). The actin cytoskeleton and

actin-regulatory proteins play vital roles in generating the forces and shape changes

required for cell movement.

Actin

Actin is a globular protein that is separated into two lobes by a cleft that forms the

ATP-binding site (Bettinger et al., 2004). ATP-bound actin monomers (globular or

G-actin) can assemble into filaments filamentouss or F-actin), associated with the

hydrolysis of ATP. Actin filaments are composed of two strands that twist around one

another to form a double right-handed helix. These filaments are further organized by

accessory proteins into diverse structures that carry out actin's cytoplasmic functions.

There are three isoforms of actin in higher eukaryotes; a-actin is muscle specific, whereas

p- and I-actin are found in all cell types (Bettinger et al., 2004).

Actin assembly from G-actin to F-actin occurs in a three-step process. The first

step is also the rate-limiting step (called nucleation or the lag phase). At least three actin

monomers need to aggregate for nucleation to begin. After the initial setup, the growth

or elongation phase occurs as subunits rapidly add to the nucleated filament ends. The

final step is the steady-state phase, when a balance exists between the rate of new

subunits adding to the filament ends and the rate of subunits leaving the ends. A value

known as the critical concentration designates the concentration of free subunits left in









solution once the steady state is reached. Below this value actin will depolymerize and

above the critical concentration it will polymerize (Oosawa and Asakura, 1975).

Once assembled into a filament, actin has two distinct ends: a barbed end and a

pointed end. The pointed end is known historically for the arrow-like appearance of

myosin heads bound along the filament (Alberts et al., 2002). The barbed end is also

called the plus end, and is the more dynamic of the two ends; meaning that monomers

will grow and depolymerize faster at this end than at the pointed or minus end.

Actin-Regulatory Proteins

A myriad of proteins are dedicated to actin regulation. These include proteins that

nucleate, bind monomers, side bind, bundle/cross-link, cap and sever actin (Ayscough,

1998). Actin binding-proteins are also found in the nucleus (Bettinger et al., 2004). I

focused on the capping and severing functions of actin regulatory proteins. When one

end of the actin filament is capped, no further polymerization or depolymerization occurs

at that end. A protein that severs binds alongside the actin filament and inserts itself

between adjacent subunits to weaken the noncovalent bonds of the filament, until it

severs and binds to one of the barbed ends (Ferrary et al., 1999).

Both capping and severing abilities play essential roles in cell motility. When a

protein severs an actin filament, new ends are created as old filaments are dismantled.

Consider a macrophage in fierce pursuit of a bacterium. Inside the macrophage is an

actin network that is continuously chopped up by severing proteins and redistributed to

form new positions that the macrophage must assume to catch its bacterial prey.

A model that illustrates the importance of capping is cellular necrosis. When a cell

dies, both G-actin and F-actin pour into the extracellular space. Conditions in this space

(ionic strength, pH, and temperature, etc.) are conducive for rapid polymerization of the









actin monomer pool which leads to increased viscosity and potential damage to the

surroundings. Proteins that cap actin stop this elongation, reduce viscosity, and thwart

uncontrolled polymerization (Burtnick et al., 1997).

Gelsolin, an 82 kDa protein that contains six domains and has the ability to cap and

sever actin filaments, was discovered in 1979 by Yin and Stossel. Gelsolin along with

related proteins including villin, severin, adseverin, fragmin and CapG, constitute the

gelsolin superfamily.

Origins of the Name CapG

My study focused on CapG. This gelsolin family member was discovered in 1986

by Southwick and DiNubile. CapG has also been called macrophage capping protein

(MCP), gCap39, and Myc basic motif homolog-I (Mbhl). Then in 1994 under a

suggestion from the Genome Data Base organizers at Johns Hopkins University Medical

School, researchers agreed on the name CapG. The "Cap" denotes the protein's ability to

cap the barbed ends of actin filaments, but not to sever them. The "G" signifies that the

derived amino acid sequence most closely resembles gelsolin (Mishra et al., 1994). Due

to this relatedness, gelsolin will be compared alongside CapG for most of the discussion.

Characterizing CapG

CapG has a molecular weight of 38 kDa. There are three 14 kDa repeat

subdomains, as compared to gelsolin which contains six of these 14 kDa repeats (Mishra

et al., 1994). CapG has a stokes radius of 3.0 nM, and its isoelectric point is 6.6,

migrating as a single polypeptide (Southwick and DiNubile, 1986).

According to Mishra et al. (1994) the CapG gene is 16.6 kb with 10 exons and

9 introns. The open reading frame is 6.9 kb with 9 exons, 8 introns, and 3 splice sites that









are nearly identical to the human gelsolin gene. The proximal short arm of chromosome

2 houses the CapG gene.

Evolution/Sequence Comparison

In 1994 Mishra et al. asked whether gelsolin and/or villin may have arisen from

CapG gene duplication. However the pathway to this evolutionary jigsaw appears to be

more complex than simple CapG gene duplication. One clue to the complexity of this

issue is derived from sequence comparisons. When CapG is compared to other gelsolin

family proteins, the sequence identity varies depending on the region of the protein as

well as its origin (i.e., mammalian vs. other). The first three domains of mammalian

gelsolin show a 49% sequence identity to CapG. However, the last three domains are

only 16% identical, thus casting doubt on the possibility of CapG gene duplication

(Dabiri et al., 1992). Mammalian villin also showed this sequence identity discrepancy

with a 41% identity in the first three domains, and only a 10% identity in the last three

domains (Dabiri et al., 1992). Other family members with three domains include fragmin

(Physarum polycephalum) and severin (Dictyostelium discoideum), that show sequence

identity to CapG of 30% and 25%, respectively (Dabiri et al., 1992).

CapG Sequence, Structure and Function

CapG possesses three domains. According to Yu et al. (1991) domain I is

gelsolin's equivalent for actin monomer binding. CapG domain I is dependent on Ca2

for actin binding where as gelsolin does not share this dependency. Gelsolin has 6

domains, referred to as G1-G6. In gelsolin's N-terminal half there is an actin

side-binding site (domains II and III), and a monomer binding site (domain I), but domain

I and half of domain II are required for severing actin filaments (Yu et al., 1991).









In 1991 Prendergast and Ziff probed for candidate factors to interact with the

c-Myc oncoprotein. They found CapG and therein a distantly related

basic/helix-loop-helix (B/HLH) DNA-binding motif. The real catch was the potential

nuclear localization signal dubbing CapG a nuclear protein. In 1993 this fact was

solidified by Onoda, Yu and Yin who discovered that CapG was indeed both a nuclear

and cytoplasmic protein. Another 1993 paper by Onoda and Yin showed that CapG

could be phosphorylated and that this phosphorylated form (enhanced by okadaic acid) of

CapG was abundant in the nucleus, perhaps providing some sort of subcellular regulation.

CapG's size of 38 kDa places it on the border line of being actively transported or

passively diffused into the nucleus which becomes hampered and inefficient as protein

size approaches 20-40 kDa (Van Impe et al., 2003). They also reported that CapG did

not possess the nuclear localization signal typified by other nuclear actin-binding proteins

(i.e., supervillin and zyxin). Severin and fragmin were found to contain Rev-like nuclear

export signals in their N-terminus which controlled their passage from the nucleus to

cytoplasm.

In terms of function, CapG has the ability to cap the barbed ends of actin filaments.

It should again be noted that CapG unlike its other family members is not capable of

severing actin filaments.

Regulation of CapG

Capping activity in CapG requires micromolar concentrations of Ca2+. CapG can

dissociate from actin filament ends readily either by a decrease in Ca2+ levels to

submicromolar concentrations (Southwick and DiNubile, 1986) or by increasing

phosphoinositide 4,5-bisphosphate concentrations (Yu et al., 1990). Gelsolin uncapping









requires both increases in phosphoinositides and a decrease in Ca2+ concentrations (Yu et

al., 1990).

Cellular Distribution

With the exception of platelets, CapG is found in nearly all cells usually at low

concentrations of approximately 0.05% of the total protein. However, CapG is one of the

most abundant cytoplasmic proteins in macrophages and dendritic cells, where it

accounts for 0.9-1% of the total cytoplasmic protein (Dabiri et al., 1992; Parikh et al.,

2003). Yu et al. (1990) also purified CapG from human plasma where it appeared to be a

minor component. Gelsolin also has a broad tissue distribution and is found in platelets

as well as smooth and skeletal muscle cells (Dabiri et al., 1992).

Membrane Ruffling

Loss of CapG affects cell movement (Witke et al., 2001). Macrophages from

CapG knockout mice demonstrate decreases in phagocytosis and membrane ruffling. In

2003, Parikh et al. once again studied CapG knockout mice with their impaired

membrane ruffling and found the null mice to be more susceptible to infection by the

food borne pathogen Listeria monocytogenes, but demonstrated normal susceptibility to

Salmonella enterica Serovar Typhimurium infection. This suggested that patients with

unexplained listeriosis may be suffering from a CapG deficiency, although no current

CapG defects in humans have been described (Parikh et al., 2003).

Mutations to CapG

In 1995 Southwick reported a series of gain-of-function mutations to CapG which

rendered the protein capable of severing actin filaments. First the sequences of CapG,

gelsolin, villin, severin and fragmin were scrutinized for consensus sequences likely to

confer severing function (Figure 1-1). Two CapG sequences were found to be divergent.









Thus mutations to convert the divergent stretches to gelsolin sequences were performed

and a gain-of-function resulted. The resultant mutant CapG 124GFKHV128 and

84LDDYLGG90 still did not sever as well as gelsolin (gelsolin required one-fiftieth the

concentration to cause severing comparable to the 124GFKHV128 and 84LDDYLGG90

mutant). The LDDYLGG region is known to be the central region of a long a-helix that

interacts with the subdomain 1 and 3 cleft of the actin monomer (Southwick, 1995).

Further mutations were performed to CapG 124GFKHV128 and 84LDDYLGG90 again

as dictated by sequence divergence in other gelsolin family members. Amino acids

129-137 of CapG were made identical to gelsolin. The new mutant CapG

124GFKHVVPNEVVVQR137 and 84LDDYLGG90 proved to be a more potent severer than

the original gain-of-function by 3-4 fold (Zhang et al., unpublished).

Severing Mutant Mutations

My contribution to understanding how CapG's structure relates to its kinetic

functions was to perform truncation and addition mutations to the already mutated CapG

124GFKHVVPNEVVVQR137 and 84LDDYLGG90 (here called CapG severing mutant).

One addition and two deletions were made in sets of three amino acid changes to the

linker region between domains I and II that is believed to hold spatial significance to the

severing ability previously bestowed upon this mutant. It was determined that three

amino acid changes would be sufficient to alter this linker's length, and might be

expected to alter CapG severing mutant's function as reflected by kinetic analysis

experiments.










Mutations performed in my studies were as follows:


* CapG severing mutant

* Deletion of -KHV

* Addition of +AAA

* CapG severing mutant

* Deletion of -GGV


15KYQEGGVESGFKHVV


115KYQEGGVESGF


115KYQEGGVESAAAGFKHVV

115KYQEGGVESGFKHVV

115KYQE ESGFKHVV


I postulated that these mutations would not alter the ability of CapG severing

mutant to cap the barbed ends of actin filaments. However, given the complexity of the

severing mechanism, I proposed that the mutations would modify the proteins ability to

sever actin filaments.


A cap G
Gelsolin
Villin
Severin
Fragnin

B Cap G
Gelsolin
Villin




C Cap G

Villin
Severin
Fragi-dn


i t. F K V 58


SV L H f l
A I F V 'IN5 1114
IYA I Y rT KI

SA YC K 1. G, 120
LD LAGROIL,aXJL MK 191

E L,4 'E SG r F[]EK IfT APAAIK
K A S F WVJ P N E V V V Q
St.V A S I& K*VE T N S3 Y DVQ
S b f;E S 4 F N fl: K PTE Y K P -
D G :E T .u UE AD KYR -


Figure 1-1. Sequence comparisons of CapG, gelsolin, villin, fragmin, and severin. A)
amino acid sequences were compared within the region previously suggested
to be important for severing. The shaded areas show regions of high identity.
Two regions (in B and C) revealed deviation of the CapG sequence (boldface
letter in unshaded blocks from the consensus sequences shared by the severing
proteins. Gaps are designated by dashes. The region of identity to the a- and
P-actin sequence in underlined in B. The region highly conserved among the
severing proteins, but different in CapG, is underlined in C. (Southwick, F.S.
1995. Gain-of-function mutations conferring actin-severing activity to human
macrophage cap G. J. Biol. Chem. 270:45-8. Figure 1, page 46.)














CHAPTER 2
MATERIALS AND METHODS

Site-Directed Mutagenesis

Human CapG cDNA was cloned into pET12a at Ndel and Sail polylinker sites as

previously described by Dabiri et al. (1992). Mutants were generated by Stratagene's

QuickChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the

manufacturer's instructions. PCR melting, annealing, and elongation temperatures used

were 95, 55, and 68C respectively.

Protein Expression and Purification

Clones in BL21(DE3) were grown in 1L LB at 37C containing 50 tg/ml

carbenicillin to an A600 = 0.6 and induced with 0.5 mM isopropyl-l-thio-/f-D-galactoside

(IPTG). Upon induction CapG severing mutants -KHV and -GGV were grown for 3 h at

25C. CapG severing mutant +AAA remained at 37C for 3 h post-induction. To harvest

cells, centrifugation was carried out at 5,000 X g for 5 min at 4C. Pellets were

resuspended in 10 ml lysis buffer (10 mM Imidazole, 1 mM EGTA, 1 mM DTT, IX mini

cocktail protease inhibitor tablet from Roche Applied Science, Mannheim, Germany) and

then freeze thawed three times to lyse cells. 100 .g/ml lysozyme and 10 .g/ml DNaseI

was added and samples shaken for 30 min at 25C before sonication at 80% power for

3 min. The cell lysates were then centrifuged at 40,000 X g for 30 min at 4C and

resultant supernatant and pellet fractions were analyzed on Coomassie Blue stained

SDS-polyacrylamide gels.









The supernatants were loaded onto DEAE ion-exchange columns (Amersham

Biosciences). If necessary samples were concentrated in a 9 kD Apollo 20 ml high

performance centrifugal spin concentrators (Orbital Biosciences, LLC, Topsfield, MA)

for 15 min at 170,000 X g, 4C, and repeated until a volume of 5 ml was attained before

being loaded onto a HiPrep desalting column (Amersham Biosciences).

Coomassie Blue stained SDS-polyacrylamide gels were employed to analyze the

proteins purity. Pure fractions were pooled and stored in 30% ethylene glycol. The

method of Bradford was used to obtain protein concentrations using BSA standards

(Bradford, 1976).

CapG severing mutant was grown and harvested following the same protocol as the

mutants except that CapG severing mutant was grown at 37C for 2 h post-induction.

Actin Purification

Actin was purified from skeletal muscle (rabbit, porcine, chicken) and conjugated

to N-(1-pyrenyl) iodoacetamide at actin's Cys 374 as previously described (Young et al.,

1990). Briefly, actin was made into an acetone powder (Spudich and Watt, 1971; Pardee

and Spudich, 1982) by mincing muscle and extracting it with 1L 0.1 M KC1, 0.15 M

potassium phosphate, pH 6.5 before being filtered through cheesecloth, stirred in 2L

0.05 M NaHCO3 and then cheesecloth filtered again. This extraction was followed by the

addition of 1L 1 mM EDTA, pH 7.0 and then two extractions of 2L of distilled H20. The

last five extractions were performed with 1L acetone. The filtered residue was air dried

in glass evaporating dishes overnight in a fume hood. The resulting acetone powder was

extracted in Buffer-G (5 mM Tris, 0.1 mM CaC12, 0.2 mM ATP, 0.2 mM DTT, 0.01%

Sodium Azide, pH 8.0) and filtered through cheese cloth before being ultracentrifuged for

40 min at 4C, 28,000 X g. The supernatant was slowly stirred at room temperature for









1 h while adding 2 ul/ml MgC12 and 52 mg/ml KC1 to polymerize the actin, and followed

by centrifugation for 3 h at 4C, 49,300 Xg. The actin pellet was resuspended in a

minimal volume of Buffer-G and dialyzed against fresh Buffer-G for 3 days to

depolymerize the filaments. Residual F-actin was removed by ultracentrifugation for

45 min at 4C, 49,300 X g. The supernatant was loaded onto a S-200 Column

equilibrated with Buffer-G and eluted with Buffer-G. Purified monomeric actin was

labeled with pyrene, N-(1-pyrenyl) iodoacetamide, according to standard procedures

(Kouyama and Mihashi, 1981). The critical concentration was determined before each

assay and found to be between 0.01 tM and 0.08 aM. (Cooper et al., 1983).

Kinetic Assays

Subcritical Actin Monomer Fluorescence Assay

Increasing amounts of protein were added in a glass cuvette with monomeric actin

just below the critical concentration at 300 nM G-Actin in S1 buffer (10 mM imidazole,

100 mM KC1, 0.4 mM MgC12, 0.1 mM CaC12, 0.1 mM ATP, 0.5 mM DTT, pH 7.6). The

solution was mixed 5X after addition of protein to ensure the formation of protein-actin

complexes. These complexes are weakly fluorescent due to the conformation change of

the actin molecule labeled with pyrene. Fluorescent intensity of these protein-actin

complexes was measured and once the intensity values stabilized, additional CapG

mutant protein was added to the solution, mixed 5X, and the intensity was measured

again. The final intensity values were plotted against protein concentration.

Fluorescence was monitored on a HORIBA Jobin Yvon FluoroLog 3 spectrofluorometer

(Edison, NJ) with Xx = 366 nm and Xem= 385 nm while maintaining a slit length at

5.0 mm. Data was collected using DataMax Software (HORIBA Jobin Yvon Edison,

NJ).









Monomer Sequestration Assay

2 tM Actin was polymerized in S1 buffer (10 mM imidazole, 100 mM KC1,

0.4 mM MgC12, 0.1 mM CaC12, 0.1 mM ATP, 0.5 mM DTT, pH 7.6) using gelsolin

capped F-actin nuclei, molecular ratio 1:20 (gelsolin to actin, final concentration of

F-actin seeds of 0.1 .iM). Assembly rates of the pointed end were measured in the

presence of increasing concentrations of capping protein, 3 [tM G-actin, and 200 tl 2XP

buffer (20 mM imidazole, 0.2 mM KC1, 4 mM MgCl2, 2 mM ATP, 2 mM DTT, pH 7.4).

The capping protein sequestered monomers away from the G-actin pool resulting in a

decrease in fluorescence of pointed end filament assembly. Fluorescent intensity was

observed with respect to time on the FluoroLog 3 (same settings as described before).

Capping Assay

2 tM Actin was allowed to polymerize overnight in S1 buffer (10 mM imidazole,

100 mM KC1, 0.4 mM MgCl2, 0.1 mM CaC12, 0.1 mM ATP, 0.5 mM DTT, pH 7.6) at

25C in the dark. F-Actin was diluted 1:20 below the critical concentration in S1 buffer

to 100 nM in a glass cuvette. F-Actin was mechanically sheared 5X with an extended

length p200 pipette tip (Fisher Scientific, Suwanee, GA) to create new barbed and

pointed ends that rapidly depolymerized as shown by a decrease in fluorescent intensity.

Increasing amounts of capping protein were added to the buffer which capped the barbed

ends of the actin filaments and retained fluorescent intensity. Fluorescence was

monitored over time on the FluoroLog 3 (spectrofluorometer settings as described

before).

Severing Assay

2 tM Actin was allowed to polymerize in S1 buffer (10 mM imidazole, 100 mM

KC1, 0.4 mM MgCl2, 0.1 mM CaCl2, 0.1 mM ATP, 0.5 mM DTT, pH 7.6) using gelsolin










capped actin filament molar ratio 1:200 (gelsolin to actin). F-Actin was diluted 1:20

below the critical concentration in S1 in buffer to 100 nM in a glass cuvette and mixed

gently 5X to avoid shearing filaments (and keep fluorescence at a steady intensity) before

adding capping protein while mixing another 5X. Severing proteins create new barbed

and pointed ends that depolymerize below the critical concentration resulting in

decreased fluorescent intensity. Fluorescence was observed with respect to time on the

FluoroLog 3. Spectrofluorometer settings were the same.

Table 2-1. Primer design for CapG severing mutants.
+AAA-foward
A A A__________
119 120 121 122 123 124 125 126 127 128 129
119 120 121 122 123 124 125 126 127 128 129 130 131 132
GGT GGT GTG GAG TCA GCG GCG GCG GGA TTT AAA CAC GTG GTT
G G V E S A A A G F K H V V
Gly Gly Val Glu Ser Ala Ala Ala Gly Phe Lys His Val Val
+AAA-reverse
AAC CAC GTG TTT AAA TCC CGC CGC CGC TGA CTC CAC ACC ACC


-KHV-forward (delete K126 H127 V128)
___ ___ ______________A A A______________
119 120 121 122 123 124 125 126 127 128 129 130 131 132 133
GGT GGT GTG GAG TCA GGA TTT AAA CAC GTG GTT CCG AAC GAA GTT
G G V E S G F K H V V P N E V
Gly Gly Val Glu Ser Gly Phe Lys His Val Val Pro Asn Glu Val
-KHV-reverse
AAC TTC GTT CGG AAC CAC GTG TTT AAA TCC TGA CTC CAC ACC ACC


-GG"V--fnrward (delete C119 GC120 V121)


A A A
112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127
CGG GGC CTC AAG TAC CAG GAA GGT GGT GTG GAG TCA GGA TTT AAA CAC
R G L K Y Q E G G V E S G F K H
Arg Gly Leu Lys Tyr Gin Glu Gly Gly Val Glu Ser Gly Phe Lys His


-GGV-reverse
GTG TTT AAA TCC TGA CTC TTC CTG GTA CTT GAG GCC CCG














CHAPTER 3
RESULTS

Purification of CapG Mutants

All mutants were purified as outlined in Materials and Methods. CapG severing

mutant +AAA was first passed through a DEAE column and analyzed on Coomassie

Blue stained SDS-polyacrylamide gels. Fractions 42 to 61 were concentrated in a 9 kD

Apollo 20ml high performance centrifugal spin concentrator before being loaded onto a

large HiPrep desalting column. The protein was found to be of 99% and 96% purity and

collected from fractions 5-7 and 10-11, respectively (Figure 3-1A). CapG severing

mutant -GGV was found to be of 99% and 90% purity and collected from fractions 17-19

and 20-24, respectively, of a DEAE column (Figure 3-1B). CapG severing mutant

-KHV was collected from pooled fractions 32-37 from a DEAE column and found to be

of 98% purity (Figure 3-1C).

Structure-Function Analysis

Effects of Actin Monomer Binding by CapG Mutants

We first assessed the ability of the mutant protein to bind monomeric actin. A

subcritical actin monomer fluorescence assay (which operates based on change in

fluorescence intensity of a subcritical concentration of pyrenyl actin; refer to Materials

and Methods), was performed on all mutant and wild type proteins. The conformational

change in actin upon polymerization is readily detected by the sulfhydryl reagent

N-(l-pyrenyl) iodoacetamide. This probe was found to display a fluorescence spectrum

more sensitive than previously reported probes, and has since been the standard for









kinetic assessment (Kouyama and Mihashi, 1981). When pyrenyl actin undergoes

polymerization a 20 fold increase in fluorescence is observed (Kouyama and Mihashi,

1981). However, in the subcritical monomer assay where no polymerization occurs, only

a 2 to 3 fold increase in fluorescence was seen. This small rise in fluorescence reflects a

conformational change in monomeric actin as capping protein-actin complexes are

formed (Southwick and DiNubile, 1986; Young et al., 1990 and 1994).

In Figure 3-2B CapG severing mutant +AAA reaches peak fluorescence at

2000 nM. The KD (dissociation) was determined to be 1000 nM. CapG severing mutant

-GGV achieved saturation at 5000 nM (Figure 3-2C) yielding a KD of 2,500 nM. CapG

severing mutant has a higher affinity for actin monomers with a KD of 150 nM (Figure

3-2A), than the +AAA and -GGV mutants. Wild type CapG has a KD reported to be

1000 nM (Young et al., 1990).

For reasons that are unknown, and in contrast to the other mutants, the -KHV

mutant was not amenable to study using the subcritical actin monomer fluorescence

assay. To overcome this I employed a second assay known as the monomer sequestration

assay to determine the monomer binding affinity in this mutant. For this monomer

sequestration assay, gelsolin is incubated with actin (1:20) which caps the barbed end of

the actin filament leaving only the pointed ends open for elongation. G-actin is added to

the reaction cuvette above the critical concentration of the pointed end allowing

polymerization at that end. When -KHV mutant is added it cannot cap the pointed end,

therefore any reduction in the rate of pointed end assembly would result from its ability

to bind and prevent actin monomers from adding to the filament (often termed monomer

sequestration). Figure 3-2D shows that very high amounts of CapG severing mutant









-KHV are required to inhibit actin assembly. 3000 nM is required to completely

sequester actin monomers and the KD is estimated to be 1,500 nM. The affinity of this

mutant for actin monomers is markedly reduced when compared to the primary severing

mutant (Figure 3-2A). All mutants, +AAA, -GGV and -KHV displayed this weaker

binding affinity with a KD of 1,000 nM, 2,500 nM and 1,500 nM respectively.

Capping

The ability of the mutants to cap barbed ends of actin filaments was assessed via a

capping assay. In this assay, actin was diluted below the critical concentration and

mechanically sheared as described in Materials and Methods to create free barbed ends

that rapidly depolymerize. Pointed ends are also created, however, the dynamics at the

pointed end are considered negligible in comparison to the more dynamic barbed end.

Capping protein added in increasing amounts to the dilute actin will cap the barbed ends

and retain fluorescence as depolymerization halts.

In Figure 3-3A CapG severing mutant +AAA begins to slow depolymerization at

2.5 nM. The addition of 10 nM, 20 nM and 100 nM shows similar retention of

fluorescence as the +AAA mutant caps the barbed ends. Note the saturation value of

capping protein will never be a perfectly straight line due to the slight depolymerization

occurring at the pointed end. CapG severing mutant -GGV began capping monomers at

0.5 nM (Figure 3-3B). Good fluorescence retention is observable at concentrations of

12 nM.

CapG severing mutant +AAA and -GGV proved to be most similar to wild type

CapG with half maximal capping values of 5 nM and 3 nM respectively. Wild type CapG

is reported by Southwick (1995) to have a half maximal capping value of 0.5 nM. CapG

severing mutant also has a half maximal capping value of 0.5 nM (Zhang et al.,









unpublished). CapG severing mutant -KHV was found to have a much higher half

maximal capping value between 20 to 50 nM (Figure 3-3C) perhaps due to steric charges

(reviewed in Discussion section).

Severing

In order to ascertain whether CapG severing mutant's ability to sever was affected

by altering the length of the domain I-II linker, a severing assay was performed (refer to

Materials and Methods for a detailed description). Gelsolin seeded actin was diluted

below the critical concentration with care taken in mixing to avoid mechanically shearing

the F-Actin (fluorescence will stay constant). When the severing protein is added to the

reaction cuvette the filaments will sever creating new barbed and pointed ends that will

depolymerize resulting in a decrease in fluorescence.

Figure 3-4A, B, and C reveal that all CapG severing mutants +AAA, -GGV and

-KHV respectively, did not exhibit any severing activity as observed by the lack of fast

depolymerization despite addition of high amounts of protein. Figure control, CapG

severing mutant, displays this rapid depolymerization.






~4W lop

4A 25 2.5L 2-.
A B C
Figure 3-1. Protein gels of the CapG severing mutant and its triplicate mutants. A)
+AAA. B) -GGV. C) -KHV. Coomassie Blue stained SDS-polyacrylamide
gels showing purified protein migrating at 38 kDa. M is protein marker, L is
the column load, and numbers denote protein fractions.








18






A B
O o +AAA
CapG severing mutant 240000 -

240 -
230000 -

g 230 o
220000
-c U o0
220
~ 0 210000
0 E
L, 210
210 200000
.> 0
M 0)
200 -190000
0

190 180000
0 100 200 300 400 0 2 4
Concentration, nM Concentration, pM

C D o Control
A 700nM -KHV
1500nM -KHV
O -GGV 500- 1700nM -KHV
3000nM -KHV
110-
O 450 oO
100- 0
o
S70o
60 0
r- 400- 0 A









250
S0 AAA

0 80- 350 10 A
0 0 OA
U- 70- Z 0A




205


200 0 .
40, o 60 120 180 240 300 360
0 2 4 6
Concentration, pM

Figure 3-2. Monomer Binding of CapG Severing Mutant and its triplicate mutants. A)
CapG Severing Mutant. B) +AAA. C) -GGV. Increasing amounts of
capping protein were added to 300 nM G-actin in S1 buffer and mixed, and
the observed fluorescence increase plotted versus the final concentrations of
capping protein. D) Pointed-end growth rate inhibited by the presence of
increasing amounts of CapG severing mutant -KHV. Fluorescence was
monitored after the addition of 100 nM gelsolin seeded actin added to S1
buffer, capping protein, 3 [M G-actin, and 2XP buffer (refer to Materials and
Methods). Open circles denotes all components mentioned above except for
capping protein.












O Control
V 2.5nM +AAA
* 5nM +AAA
0 lOnM +AAA
* 20nM +AAA


A 100nM+




0 0 *..

0 0 O g*
0 *0s.


0
o0
o 'r


AAA


o
U
-,
0

,m
LL


V


00000


120 180
Time, s


A AAA
E o o


:
o *

a


0
0


O Control
A lOOnM-KHV
S 50nM -KHV
20nM -KHV
* lOnM-KHV


o

0 o 0
o *
o OO


ooo
120 180
Time, s


0 Control
* 0.5nM -GGV
3nM -GGV
* 12nM -GGV
A 20nM -GGV


O *0

o 0
0 *

o


AAAAAAAAAAA











OO.
00
00
oo
000000


1UUU .-
0 60 120 180
Time, s

Figure 3-3. Capping activities of the CapG Severing Mutant triplicate mutants. A)
+AAA. B) -GGV. C) -KHV. Depolymerization of the barbed end of 2 PM
F-actin ( Materials and Methods) diluted below its critical concentration to
100 nM in S1 buffer is halted by the addition of increasing amounts of
capping protein (filled symbols) as evidenced by the retention of fluorescence.

Note the saturation value of capping protein will never be a perfectly straight
line due to the slight depolymerization occurring at the pointed end. Open

circles denote the disassembly rate of 100 nM pyrene actin in S1 buffer.


600-



500-_
0


R

2400


2200

U
S2000-
U
U)
- 1800-
O
U-
1600-


S1400


1200












---- Control
5-- OnM +AAA
-- 100nM+AAA
---- 200nM +AAA
-- 200nM CapG severing mutant


Time, s


-0- Control
- 5OnM KHV
-- 75nM-KHV
-- 200nM KHV
-- 200nM CapG severing mutant


20
Time, s


--0- Control
- l10nM -GGV
-A- 5000nM -GGV
---- 6000nM -GGV
- -- 200nM CapG severing mutant


0 20 40 60
Time, s

Figure 3-4. CapG Severing Mutant triplicate mutants lack severing activity. A) +AAA.
B) -GGV. C) -KHV. Increasing amounts of capping protein was added to
gently mixed 2 atM F-actin (refer to Materials and Methods) diluted below its
critical concentration to 100 nM in S1 buffer. The depolymerization as
observed in the CapG severing mutant control (filled circles) denotes
severing. Open circles are the same concentration of pyrene actin in S1
buffer. No acceleration in the depolymerization rate was observed in mutants
indicating a lack of severing activity.














CHAPTER 4
DISCUSSION

Role of the S1-S2 Linker Length on Function

My studies were designed to explore the effects of length change of the domain I-II

linker region of the CapG severing mutant, and are based on the crystal structures of the

CapG severing mutant (Figure 4-1) and gelsolin (Burtnick et al., 2004; Zhang et al.,

unpublished). The linker region between domains I and II in both activated CapG

severing mutant and gelsolin is extended at 36A and 30A respectively (Burtnick et al.,

2004; Zhang et al., unpublished). This linker positioning is imperative to allow the

LDDYL loop steric interactions resulting in severing of the actin filament (McLaghlin et

al., 1993; Zhang et al., unpublished).



Structural Determinants of Capping and Severing

The model for CapG capping is thought to be similar to gelsolin G1-G3 capping,

which has been made possible through the efforts of X-ray crystallography, electron

microscopy (EM), and nuclear magnetic resonance (NMR) spectroscopy (McGough et

al., 2003). Figure 4-2 depicts G1 bound to subunit 1 at the very end of the actin filament

and G2 bound in an area bridging subunit 1 and 3 (McGough et al., 2003). Past studies

by Irobi and colleagues place G1 and G2 on adjacent monomers of the same long-pitch

helix of the filament. In either case, the triplicate mutants of the CapG severing mutant

did not disrupt capping activity, indicating that larger length alterations in the domain I-II

linker region may be needed to misalign domain I and impair capping function.









Severing is a more complex function. In full length gelsolin, severing is initiated

by Ca2+ which causes the protein to undergo many conformational changes (Burtnick et

al., 2004). For instance Sun, et al. (1999) reports that in the presence of Ca2+ the

extended P-sheet between G4 and G6 is broken along its interface, G6 swings out from

G4 to form new contacts with G5, and actin becomes situated into the unoccupied G6

space to create an intermolecular Ca2+ binding site coordinated by G4 and actin.

Ultimately for severing, G2-G3 attaches alongside an actin filament while the flexible

G1-G2 linker extends between two adjacent protomers on the long-pitched actin strand

(Burtnick et al., 2004). Simultaneously, in a separate direction the G3-G4 linker and G6

wrap over the filament's surface and direct G1 and G4 to their binding sites (Burtnick et

al., 2004). A concerted pincer motion of G1 and G4 imparts a steric strain so great that

enough non-covalent bonds between the actin monomers in the filament below are

weakened and the filament is severed (Burtnick et al. 2004; Sun et al., 1999). Figure 4-3

illustrates the severing process in gelsolin.

CapG and other members of the gelsolin superfamily are thought to possess the

same mechanism of attachment to the actin molecule as the gelsolin domain I and II

linking peptide, based on sequence homology (Irobi et al., 2003). Although gelsolin

severs the filament in two locations, the CapG severing mutant only severs in one

location within the actin filament because it can only coordinate three subunits. We

predicted that small changes in the domain I-II linker could affect the positioning of the

LDDYL severing loop on the actin filament. Therefore severing would be expected to be

more sensitive to alterations in the length of the linker as compared to capping.









Comparison between CapG Severing Mutant and New Mutants

To interact with actin, CapG severing mutant must first bind to one or more actin

monomers within a filament. Therefore, monomer binding was first assessed. Deleting

or adding amino acids in triplicate was detrimental to CapG severing mutants' monomer

binding affinity as evidenced by a 7-fold reduction in +AAA, a 17-fold reduction in

-GGV and a 10-fold reduction in -KHV. When compared to wild type CapG with a KD

reported to be 1000 nM (Young et al., 1990) the triplicate mutants' affinity for

monomeric actin is comparable (a KD of 1000 nM, 2,500 nM and 1,500 nM for +AAA,

-GGV and -KHV mutants respectively, Table 4-1).

In Zhang et al., unpublished, it has been postulated that CapG severing mutant

contains two actin binding sites. The addition mutant +AAA appears to also possess this

second actin binding site as evidenced by the decrease in fluorescence at very high

concentrations of the mutant protein. The deletion mutants -GGV and -KHV appear to

have a stoichiometry similar to wild type CapG with only one actin binding site, since the

fluorescence remains elevated at very high concentrations of protein.

The ability to cap was also hindered for the -KHV mutant who's half maximal

capping constant was between 20-50 nM as compared to wild type CapG and CapG

severing mutant, both with a half maximum capping at 0.5 nM. CapG severing mutant

-KHV appeared to be the odd mutant out when compared to mutants +AAA and -GGV.

This difference could be the consequence of electrostatic charges. The lysine (K),

histidine (H), and valine (V) are associated with basic, basic, and neutral charges

respectively. Whereas the other mutants addition of three neutral alanines (A) and

deletion of neutral glycines (G) and valine, would have no effect on charge but simply

affect the length of the linker region.









The most profound functional effect was on severing. Figure 4-1 helps illustrate

how alterations in length will affect what amino acid charges get placed adjacent to each

other. In mutation site II where the triplicate mutations were made, the side chains are

visible. These side chains can attract or repel the actin residues that make contact with

the CapG severing mutant during binding. Side binding of the actin filament is necessary

for severing to occur, thus the contacts between the side-binding protein and actin need to

be favorable. All triplicate mutants of the CapG severing mutant did not sever, once

again stressing the importance of both length and charge of the linker region between

domains I-II in order for severing to occur.


Table 4-1. Functional activities of CapG and its mutants
KD of 12 max Severing
Protein G-actin capping activity
(nM) (nM)
1000 0.5
CapG (Young et (Southwick, No
al., 1990) 1995)
CapG 0.5
severing 150 (Zhang Yes*
mutant et al.,
unpublished)
CapG
severing 1000 5 No
mutant
+AAA
CapG
severing 2,500 3 No
mutant -GGV
CapG
severing 1,500 20-50 No
mutant -KHV
*Half-maximal severing reported to be 30-50 nM (Zhang et al., unpublished).




















S2 domain


$3 domain SI domain


Figure 4-1. Structure of the CapG severing mutant. (Zhang, Y., S.M. Vorobiev, B.G.
Gibson, B. Hao, G. Sidhu, V.S. Mishra, E.G. Yarmola, M.R. Bubb, S.C.
Almo, and F.S. Southwick. Unpublished. A CapG gain-of-function mutant
reveals critical structural and functional determinants for actin filament
severing.)
























F.i iGl= blue violet
G2=pink
G3=green
G4=aqua
.. G5=red
G6=lime green






Figure 4-2. Possible model of filament capping by gelsolin. Actin filaments are
represented by space-filling models oriented with the minus or slow-growing
end up. Actin subunits from one long-pitch strand are colored yellow and
those from the other are colored gray. (McGough, A.M., and C.J. Staiger,
J.K. Min, and K.D. Simonetti. 2003. The gelsolin family of actin regulatory
proteins: modular structures, versatile functions. FEBS Lett. 552:75-81.
Figure 2A, page 78.)






















I

p-
_, ," '


G1 and G4 are
directed to
their binding
sites


p.


0-IE


Unr


Severing


Figure 4-3. Sequence of events during severing of actin by fully activated gelsolin.
Actin protomers shown in blue. Gelsolin subunits are multi-colored ovals.
Reprinted by permission from Macmillan Publishers Ltd: [The EMBO
Journal] (Burtnick, L.D., D. Urosev, E. Irobi, K. Narayan, and R.C. Robinson.
2004. Structure of the N-terminal half of gelsolin bound to actin: roles in
severing, apoptosis and FAF. EMBO J. 23:2713-22. Figure 2B, page 2716.),
copyright (2004)


B1
i ~i-
I, I
ri.-' d
rr

O


G2-G3
binds














CHAPTER 5
CONCLUSION

The reorganization of the actin network by many different proteins is essential for

cell movement. The role of CapG in the cell is to cap the barbed ends of actin filaments

thereby preventing further monomer growth from that end. CapG is unique among its

gelsolin family members in that it does not possess the ability to sever filamentous actin.

Severing is necessary for cellular plasticity; it erases old actin networks so that new ones

can be formed (Ferrary et al., 1999). CapG was mutated to resemble gelsolin in two

separate locations, 124GFKHVVPNEVVVQR137 and 84LDDYLGG90 to create the CapG

severing mutant (Zhang et al., unpublished). When the length of the region linking

domains I-II is altered by three amino acids, severing function is lost but capping activity

is preserved. To further investigate how length translates structurally one would need to

perform a crystallographic analysis to view the exact positioning of the affected domain

I-II linker in the triplicate mutants compared to the CapG severing mutant.
















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BIOGRAPHICAL SKETCH

Andrea Roebuck was born in Michigan and moved to Florida at the age of four.

Studies at the University of Florida (UF) began in June 1999, with a Bachelor of Science

awarded in microbiology and cell science in May 2003. Andrea continued her graduate

studies at UF to obtain a Master of Science in molecular genetics and microbiology in

May 2006.